scholarly journals The plakin domain ofC. elegansVAB-10/plectin acts as a hub in a mechanotransduction pathway to promote morphogenesis

2019 ◽  
Author(s):  
Shashi Kumar Suman ◽  
Csaba Daday ◽  
Teresa Ferraro ◽  
Thanh Vuong-Brender ◽  
Saurabh Tak ◽  
...  
Keyword(s):  
Focal Adhesions ◽  
Sh3 Domain ◽  
Target Cells ◽  
C Elegans ◽  
Cellular Processes ◽  
Src Homology 3 ◽  
Src Homology ◽  
Summary Statement ◽  
Dynamics Simulations ◽  
Spectrin Repeats

AbstractMechanical forces control many cellular processes by eliciting a mechanotransduction response in target cells. The initial steps of mechanotransduction at hemidesmosomes remain undefined in contrast to focal adhesions and adherens junctions. Here, we focus on theC. elegansplectin homolog VAB-10A, the only evolutionary conserved hemidesmosome component. InC. elegans, muscle contractions induce a mechanotransduction pathway in the epidermis through hemidesmosomes. We used CRISPR to precisely remove spectrin repeats (SR) or a partially hidden Src-homology-3 (SH3) domain within the VAB-10 plakin domain. Deleting the SH3 or SR8 domains in combination with mutations affecting mechanotransduction, or just part of SR5 shielding the SH3 domain induced embryonic elongation arrest because hemidesmosomes collapse. Notably, recruitment of GIT-1, the first mechanotransduction player, requires the SR5 domain and the hemidesmosome transmembrane receptor LET-805. Furthermore, Molecular Dynamics simulations confirmed that forces acting on VAB-10 can render the central SH3 domain, otherwise in contact with SR4, available for interaction. Collectively, our data strongly argue that the plakin domain plays a central role in mechanotransduction and raise the possibility that VAB-10/plectin might act as a mechanosensor.Summary statementCRISPR-derived deletions reveal the roles of three spectrin repeats and an atypical SH3 domain from the plakin domain of the VAB-10 hemidesmosome component in mechanotransduction duringC. elegansmorphogenesis

scholarly journals ANKRD54 preferentially selects Bruton’s Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library

PLoS ONE ◽  
2017 ◽  
Vol 12 (4) ◽  
pp. e0174909 ◽  
Author(s):  
Manuela O. Gustafsson ◽  
Dara K. Mohammad ◽  
Erkko Ylösmäki ◽  
Hyunseok Choi ◽  
Subhash Shrestha ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Sh3 Domain ◽  
Bruton’S Tyrosine Kinase ◽  
Bruton's Tyrosine Kinase ◽  
Src Homology 3 ◽  
Src Homology

scholarly journals Src-Dependent Phosphorylation of ASAP1 Regulates Podosomes

2007 ◽  
Vol 27 (23) ◽  
pp. 8271-8283 ◽  
Author(s):  
Sanita Bharti ◽  
Hiroki Inoue ◽  
Kapil Bharti ◽  
Dianne S. Hirsch ◽  
Zhongzhen Nie ◽  
...  
Keyword(s):  
Splice Variants ◽  
Phosphorylation Site ◽  
Sh3 Domain ◽  
Tyrosine Kinase Signaling ◽  
Bar Domain ◽  
Src Homology 3 ◽  
3T3 Fibroblasts ◽  
Src Homology ◽  
Nih 3T3 ◽  
Interfering Rna

ABSTRACT Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.

Interaction of Grb2 SH3 domain with UVRAG in an Alzheimer’s disease–like scenario

2014 ◽  
Vol 92 (3) ◽  
pp. 219-225 ◽  
Author(s):  
Kasturi Roy ◽  
Oishee Chakrabarti ◽  
Debashis Mukhopadhyay
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Sh3 Domain ◽  
Binding Motifs ◽  
Src Homology 3 ◽  
Src Homology ◽  
Cellular Compartmentalization

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein which participates in trafficking pathways alongside its role in signaling. Proteins important for actin remodeling and cellular compartmentalization contain SRC Homology 3 (SH3) binding motifs that interact with Grb2. While studying the Grb2–amyloid precursor protein (APP) intracellular domain (AICD) interaction in Alzheimer’s disease cell line models, it was seen that Grb2 colocalized to compartments that mature into autophagosomes. The entrapping of AICD in the Grb2 vesicles and its clearance via autophagosomes was a survival contrivance on the part of the cell. Here, we report that Grb2, when in excess, interacts with ultraviolet radiation resistance-associated gene protein (UVRAG) under excess conditions of AICD–Grb2 or Grb2. The N-terminal SH3 domain of Grb2 specifically interacts with UVRAG, unlike the C-terminal SH3 domain. This interaction helps to understand the role of Grb2 in the autophagic maturation of vesicles.

scholarly journals Switch-like Arp2/3 activation upon WASP and WIP recruitment to an apparent threshold level by multivalent linker proteins in vivo

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yidi Sun ◽  
Nicole T Leong ◽  
Tommy Jiang ◽  
Astou Tangara ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Quantitative Imaging ◽  
Threshold Level ◽  
Actin Assembly ◽  
Cellular Processes ◽  
Src Homology 3 ◽  
Filament Assembly ◽  
Tightly Coupled ◽  
Src Homology 3 Domain ◽  
Src Homology

Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites. Quantitative imaging suggested that productive actin assembly initiation is tightly coupled to accumulation of threshold levels of WASP and WIP, but not to recruitment kinetics or release of autoinhibition. These studies provide evidence that WASP and WIP play central roles in establishment of a robust multivalent SH3 domain-PRM network in vivo, giving actin assembly onset at endocytic sites a switch-like behavior.

scholarly journals αII-Spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts

2005 ◽  
Vol 388 (2) ◽  
pp. 631-638 ◽  
Author(s):  
Björn ROTTER ◽  
Odile BOURNIER ◽  
Gael NICOLAS ◽  
Didier DHERMY ◽  
Marie-Christine LECOMTE
Keyword(s):  
Binding Protein ◽  
Focal Adhesions ◽  
Membrane Skeleton ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Src Homology 3 ◽  
Src Homology 3 Domain ◽  
Src Homology

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of α- and β-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent α-spectrin subunit in non-erythroid cells, αII-spectrin, contains two particular spectrin repeats in its central region, α9 and α10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the α9-α10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the α10 repeat of αII-spectrin whereas EVL interacts with the Src homology 3 domain located within the α9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between αII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.

scholarly journals Asef2 Functions as a Cdc42 Exchange Factor and Is Stimulated by the Release of an Autoinhibitory Module from a Concealed C-Terminal Activation Element

2006 ◽  
Vol 27 (4) ◽  
pp. 1380-1393 ◽  
Author(s):  
Michael J. Hamann ◽  
Casey M. Lubking ◽  
Doris N. Luchini ◽  
Daniel D. Billadeau
Keyword(s):  
Regulatory Mechanism ◽  
Sh3 Domain ◽  
Terminal Segment ◽  
Binding Region ◽  
Exchange Factor ◽  
Src Homology 3 ◽  
Cell Metastasis ◽  
Src Homology ◽  
And Migration ◽  
Exchange Activity

ABSTRACT Asef (herein called Asef1) was identified as a Rac1-specific exchange factor stimulated by adenomatous polyposis coli (APC), contributing to colorectal cancer cell metastasis. We investigated Asef2, an Asef1 homologue having a similar N-terminal APC binding region (ABR) and Src-homology 3 (SH3) domain. Contrary to previous reports, we found that Asef1 and Asef2 exchange activity is Cdc42 specific. Moreover, the ABR of Asef2 did not function independently but acted in tandem with the SH3 domain to bind APC. The ABRSH3 also bound the C-terminal tail of Asef2, allowing it to function as an autoinhibitory module within the protein. Deletion of the C-terminal tail did not constitutively activate Asef2 as predicted; rather, a conserved C-terminal segment was required for augmented Cdc42 GDP/GTP exchange. Thus, Asef2 activation involves APC releasing the ABRSH3 from the C-terminal tail, resulting in Cdc42 exchange. These results highlight a novel exchange factor regulatory mechanism and establish Asef1 and Asef2 as Cdc42 exchange factors, providing a more appropriate context for understanding the contribution of APC in establishing cell polarity and migration.

Crystal structure of a Src-homology 3 (SH3) domain

Nature ◽  
1992 ◽  
Vol 359 (6398) ◽  
pp. 851-855 ◽  
Author(s):  
Andrea Musacchio ◽  
Martin Noble ◽  
Richard Pauptit ◽  
Rik Wierenga ◽  
Matti Saraste
Keyword(s):  
Crystal Structure ◽  
Sh3 Domain ◽  
Src Homology 3 ◽  
Src Homology

scholarly journals Properties of phagocyte NADPH oxidase p47-phox mutants with unmasked SH3 (Src homology 3) domains: full reconstitution of oxidase activity in a semi-recombinant cell-free system lacking arachidonic acid

2003 ◽  
Vol 373 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Guihong PENG ◽  
Jin HUANG ◽  
Mellonie BOYD ◽  
Michael E. KLEINBERG
Keyword(s):  
Arachidonic Acid ◽  
Nadph Oxidase ◽  
Sh3 Domain ◽  
Free System ◽  
Cell Free System ◽  
Src Homology 3 ◽  
Rich Domain ◽  
Phagocyte Nadph Oxidase ◽  
Px Domain ◽  
Src Homology

In an early step in the assembly of the phagocyte NADPH oxidase, p47-phox translocates from the cytosol to the membrane, mediated by engagement of the N-termini of two p47-phox Src homology 3 (SH3) domains with a proline-rich region (PRR) in the p22-phox subunit of cytochrome b558. In response to phagocyte activation, several serine residues in a C-terminal arginine/lysine-rich domain of p47-phox are phosphorylated, leading to changes in the conformation of p47-phox and exposure of its N-terminal SH3 domain that is normally masked by internal association with the arginine/lysine-rich domain. We report that triple alanine substitutions at Asp-217, Glu-218 and Glu-223 in a short sequence that links the tandem p47-phox SH3 domains unmasked the N-terminal SH3 domain, similar to the effects of aspartic acid substitutions at Ser-310 and Ser-328 in the arginine/lysine-rich region. Recombinant p47-phox proteins with mutations in either the linker region or the arginine/lysine-rich domain were active in the absence of arachidonic acid stimulation in a cell-free NADPH oxidase system consisting of recombinant p67-phox, Rac1–guanosine 5′-[γ-thio]triphosphate and neutrophil membranes. Supplementing neutrophil membranes with phosphoinositides or other negatively charged phospholipids markedly enhanced cell-free superoxide generation by these p47-phox mutants in the absence of arachidonic acid, to levels equivalent to those generated by wild-type p47-phox following arachidonic acid activation. This enhancement may be related to recruitment to the membrane of p47-phox mediated by a novel secondary phox homology (PX) domain binding site that broadly recognizes phospholipids. No specific enhancement by specific phosphorylated phosphatidylinositols was found to suggest a dominant role for the p47-phox primary PX domain binding site. Truncated p47-phox S310D S328D lacking the C-terminal PRR was inactive in the cell-free system without arachidonic acid, but was fully active with arachidonic acid. This suggests that activation of NADPH oxidase in an arachidonate-free cell-free system requires association of the p47-phox C-terminal PRR with the p67-phox C-terminal SH3 domain.

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